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Development of a loop-mediated Isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus

DOI: 10.1186/1471-2180-8-163

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The assay provided markedly more sensitive and rapid detection of V. parahaemolyticus strains than conventional biochemical and PCR assays. The assay correctly identified 143 V. parahaemolyticus strains, but did not detect 33 non-parahaemolyticus Vibrio and 56 non-Vibrio strains. Sensitivity of the LAMP assay for direct detection of V. parahaemolyticus in pure cultures and in spiked shrimp samples was 5.3 × 102 CFU per ml/g (2.0 CFU per reaction). The sensitivity of the LAMP assay was 10-fold more sensitive than that of the conventional PCR assay. The LAMP assay was markedly faster, requiring for amplification 13–22 min in a single colony on TCBS agar from each of 143 V. parahaemolyticus strains and less than 35 min in spiked shrimp samples. The LAMP assay for detection of V. parahaemolyticus required less than 40 min in a single colony on thiosulfate citrate bile salt sucrose (TCBS) agar and 60 min in spiked shrimp samples from the beginning of DNA extraction to final determination.The LAMP assay is a sensitive, rapid and simple tool for the detection of V. parahaemolyticus and will facilitate the surveillance for control of contamination of V. parahaemolyticus in seafood.Vibrio parahaemolyticus is a marine seafoodborne pathogen causing gastrointestinal disorders in humans [1,2]. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are known as major virulence determinants of V.parahaemolyticus [3]. This bacterium is widely present in estuarine, marine, and coastal environments throughout the world [1,2]. Therefore, ingestion of raw or undercooked seafood contaminated with V.parahaemolyticus is risk factors in humans [1,2].Most V.parahaemolyticus isolates from the environment do not produce TDH or TRH. Virulent strains of V. parahaemolyticus are usually found together with larger populations of avirulent strains in the environment [1,2,4,5]. The similarity in growth kinetics of the virulent and avirulent strains is a major obstacle for selective dete

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